Abl is a Caveolin Kinase

To determine initially whether Abl phosphorylates caveolin, a fibroblast cell line expressing a temperature-sensitive form of v-Abl was used [76]. A temperature-sensitive form of v-Abl was necessary because constitutively active v-Abl leads to loss of expression of both caveolin-1 and -2 after only a few days [76,77]. (Expression of v-Src also causes down-regulation of caveolin [60,77]; however, this down-regulation is secondary to cellular transformation, and is not caused by phosphor-ylation of caveolin [76].) Caveolin-1 was one of the strongest phosphotyrosine signals detected in these cells after activation of the kinase. Caveolin-1 was also one of the most prominent phosphoproteins detected in primary human fibroblast cells overexpressing c-Abl. Caveolin-2 co-immunoprecipitated with caveolin-1, and was also phosphorylated under both of these conditions. Abl can directly phosphorylate caveolin-1 in vitro and in a yeast expression system [76,78]. The phosphorylation of caveolin-1 by Abl required Tyr14 and did not occur on a fusion protein in which this residue was changed to phenylalanine, indicating that the consensus Abl phosphorylation site is the only site in caveolin-1 phosphorylated by Abl.

To verify that Abl phosphorylates caveolin, fibroblast cell lines derived from an Abl knockout mouse (Abl-/-) were utilized. The same cells reconstituted with Abl served as controls (Abl+). Although, insulin does not stimulate caveolin phosphorylation in fibroblasts, caveolin is phosphorylated in response to oxidative stress in these cells [28,29]. Oxidative stress activates Abl [79,80]; therefore, oxidative stress-induced phosphorylation was a good system initially to test the requirement for Abl in caveolin phosphorylation [32].

Abl was required for oxidative stress-induced caveolin phosphorylation. Caveolin was not phosphorylated in response to oxidative stress in the Abl-/- cells, but phosphorylation was restored in the Abl+ cells. In contrast to caveolin, many other proteins were phosphorylated on tyrosine in the Abl-/- cells, indicating that these cells still respond to oxidative stress, but that phosphorylation of Abl substrates is specifically lost. The only site of tyrosine phosphorylation of caveolin-1 in response to oxidative stress is Tyr14, the consensus Abl phosphorylation site. Therefore, expression of Abl is necessary for phosphorylation of caveolin-1 on tyrosine in response to oxidative stress, and the only site of tyrosine phosphorylation is a consensus Abl site. Additionally, overexpression of Abl is sufficient to induce cav-eolin phosphorylation in some cell types, and purified Abl phosphorylates caveolin at Tyr14 in vitro. These data indicate that c-Abl is also a stress-induced caveolin-tyrosine kinase.

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